Protein secretion across membranes is one of the most important and complex physiological processes in growing cells. The main aim of this proposal is to analyze the molecular details of this process in vitro, specifically translocation of proteins other than lipoproteins into bacterial membrane vesicles involving signal peptidase I. The elucidation of the mechanism of protein translocation will combine genetic manipulation and biochemical studies in a well-established in vitro system for the translocation of Escherichia coli alkaline phosphatase and OmpA into inverted cytoplasmic membrane vesicles to analyze the roles of ATP hydrolysis, membrane proteins and soluble cytoplasmic factors in protein translocation. Building on the recent demonstration of the involvement of SecY/PrlA, SecA, SecB and other protein factors in protein translocation, purified factors and antibodies will be used to define their roles in the process. Exploring the physiological implication of recent observations that SecA can be phosphorylated and dephosphorylated and that a protein factor SecI inhibits protein translocation, we will test the hypothesis that the ATP requirement may be due to transient phosphorylation and dephosphorylation of SecA required for the process of protein transit through the membranes, and that SecI regulates the flow of protein translocation. To determine the functions of SecA in the membranes, the components involved in the phosphorylation and dephosphorylation will be identified and characterized. Chemical crosslinking with bifunctional reagents, followed by immunoprecipitation, and partial reconstitution with membrane vesicles and liposomes will be used to identify the components involved with SecA and SecY/PrlA in protein translocation. The protein factors that are involved in protein translocation will be purified, the partial amino acid sequence will be determined to search for homology with known proteins, and if novel, the genes will be cloned. The roles of these factors on the translocation competency of precursor molecules will be examined. An understanding of mechanisms discovered in bacteria is likely to have significant implications for secretion of proteins by human cells and should have important practical implications for the medically important proteins with the use of recombinant DNA in bacteria. Furthermore, understanding the secretion of microbial toxins is also important to medical bacteriology.